Mechanisms of Ageing and Development 111 (1999) 89 – 95 www.elsevier.com/locate/mechagedev Role of superoxide, NO and oxygen in the regulation of energy metabolism and suppression of senile diseases M. Inoue *, M. Nishikawa, E. Kasahara, E. Sato Department of Biochemistry, Osaka City Uni6ersity Medical School, Abenoku, 1 -4 -3 Asahimachi, Osaka 645, Japan Received 26 May 1999; accepted 19 July 1999 Abstract Although nitric oxide (NO) rapidly reacts with molecular oxygen under air atmospheric conditions, thereby losing its biological functions, the lifetime of this gaseous radical increases under physiologically low intracellular oxygen tensions. To understand the pathophysiological roles of NO and related molecules in aerobic life, we analyzed the effect of oxygen tensions on the NO-dependent processes in resistance arteries, isolated mitochondria, intact cells and enteric bacteria. Kinetic analysis revealed that NO enhanced the generation of cGMP and induced vasorelaxation of resistance arteries more potently under physiologically low oxygen tensions than under hyperbaric conditions. NO reversibly inhibited the respiration of isolated mitochondria, intact cells and Escherichia coli; the inhibitory effect was more marked under hypoxic conditions than under hyperbaric conditions. Kinetic analysis revealed that NO has pivotal action to increase arterial supply of molecular oxygen for the generation of ATP in peripheral tissues and to suppress energy production in mitochondria and cells in an oxygen-dependent manner. These functions of NO are enhanced by decreasing oxygen tension in situ and suppressed by locally generated superoxide radicals. Thus, cross-talk of NO, superoxide and molecular oxygen constitutes a supersystem by which the energy metabolism in cells and tissues is beautifully regulated in a site-specific manner depending on the relative concentrations of these three radical species. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Energy metabolism; Senile diseases; Superoxide; Nitric oxide; Oxygen * Corresponding author. Tel.: +81-6-645-3720; fax: +81-6-645-3721. E-mail address: inoue@med.osaka-cu.ac.jp (M. Inoue) 0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 6 8 - 8 90 M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95 1. Introduction It has been postulated for a long time that human aging undergoes with the aging of cardiovascular system. In fact, in addition to neoplastic diseases, vasogenic injury of heart and brain is one of the major cause of death in aged patients. Therefore, it is critically important to know age-related changes in the metabolism of various compounds which affect the function of vascular wall cells. Response of a tissue is feed-back regulated principally by its critical functions. Because the most important function of arteries is to supply oxygen to peripheral tissues for energy production, arterial tonus, which regulates blood pressure, might be determined by the metabolism of oxygen and related compounds, including reactive oxygen species. Based on such a concept, we studied the dynamic aspects of superoxide and nitric oxide (NO) metabolisms in and around vascular walls and isolated cells and mitochondria. The present work describes the critical role of cross-talk of NO, superoxide and molecular oxygen in the regulation of the circulatory status and energy metabolism in aerobic organisms. 2. Role of cross-talk of NO, superoxide and molecular oxygen The circulatory status of animals is regulated principally by the coordination of cardiac output and arterial resistance. Arterial resistance is regulated predominantly by vascular endothelial cells and autonomic nervous systems. Endothelial cells are the most important regulators for the contraction and relaxation of vascular smooth muscle cells. The presence of some depressor compound(s) with unstable nature has been known for many years and is named as endothelium-derived-relaxing factor (EDRF) (Furchigott and Zawadzki, 1980). NO and/or its metabolite(s) in and around resistance arteries were first recognized as EDRF (Ignarro et al., 1987). Vascular endothelial cells have two enzymes, xanthine oxidoreductase and nitric oxide synthase (eNOS), which generate superoxide and NO, respectively. Due to its gaseous nature, the intracellularly synthesized NO rapidly diffuses out of cells, enters into smooth muscle cells, binds to heme-containing proteins, such as guanylate cyclase, thereby modulating cellular metabolism and functions. Binding of NO to guanylate cyclase activates the enzyme, increases cGMP levels in smooth muscle cells and thereby decreasing vascular tonus. This system is critically important for regulation of the circulatory status that determines the amounts oxygen required for ATP synthesis in peripheral tissues. The lifetime of NO has been believed to be extremely short (B8 s). However, most in vitro experiments have been performed under air atmospheric conditions where the oxygen tension ( 220 mM) is significantly higher than that of in vivo concentrations in and around cells and tissues (0.1 25 mM). We recently reported that the lifetime of NO is significantly longer under physiologically low oxygen tensions than in air atmospheric conditions (Inai et al., 1996; Nishikawa et al., 1996, 1997; Takehara et al., 1996; Nishikawa et al., 1998). Fig. 1 shows the effect of NO on arterial resistance under different oxygen tensions. To maintain cellular M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95 91 levels of ATP, isolated tissues were generally incubated in a medium infused with pure oxygen. When infused with pure oxygen, its concentration in a medium reached as high as 690 mM. Under such unphysiologically hyperbaric conditions, NO exhibited no appreciable action to induce arterial relaxation (Takehara et al., 1999). However, when oxygen tension in the medium was decreased to physiologically low levels, the same dose of NO exhibited a significantly stronger effect than under hyperoxic conditions. This observation suggests that the EDRF action of NO in and around resistance arteries differs significantly depending on the local concentrations of oxygen and is potentiated by hypoxia and anoxia. This reaction favors the adaptive reaction by which oxygen is efficiently supplied to hypoxic tissues. Thus, cross-talk of NO and molecular oxygen might play a critical role in the regulation of the circulatory status of a tissue (Fig. 2). It should be noted that vascular endothelial cells contain xanthine oxidase, which generates superoxide radicals and that NO also reacts with superoxide radicals by a diffusion-limited mechanism. Thus, if the rate of production of superoxide and/or NO is increased in and around endothelial cells, interaction of the two radicals might strongly affect the EDRF action of NO. In fact, targeting SOD to vascular endothelial cells specifically increased cGMP levels in arterial walls and decreased the blood pressure of hypertensive animals with genetic and nongenetic etiology (Skoog et al., 1992). These observations indicate that cross-talk of superoxide, NO and molecular oxygen might constitute a supersystem by which oxygen delivery for ATP synthesis is regulated. This supersystem might also underlie the pathogenesis of hypertension and shock. Fig. 1. Oxygen-dependent relaxation of aortic rings by NO depending on the concentration oxygen in a medium. The EDRF action of NO increases with the decrease in oxygen tension. The amounts of cGMP generated by NO-stimulated arteries also increased with concomitant decrease in oxygen tensions. 92 M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95 Fig. 2. Cross-talk of NO and oxyradicals regulates arterial resistance. Targeting superoxide dismutase to vascular endothelial cells selectively normalizes the blood pressure of hypertensive rats. This effect is due to dismutation of arterial superoxide, thereby the lifetime of NO is increased and their relaxation enhanced. Cross-talk of nitric oxide, superoxide and molecular oxygen determines the tonus of resistance arteries. Other factors, such as GSH and related thiols, in and around arterial walls, also modulate the EDRF action of NO. 3. Role of cross-talk of superoxide and nitric oxide in energy metabolism It should be noted that the reaction of NO with superoxide generates peroxynitrite. Hence, local concentrations of NO would be decreased if superoxide production were increased. In fact, targeting SOD to vascular endothelial cells markedly increased cGMP levels in arterial walls and decreased the blood pressure, particularly under pathological conditions (Inoue et al., 1990). Because NO has high affinity for heme proteins, it forms dissociable complexes with various hemeproteins, such as electron transport systems in mitochondria. Thus, NO inhibited the respiration of mitochondria in a reversible manner (Fig. 3). The inhibitory action of NO was stronger at low oxygen tension than at its high concentration. It is known that inhibition of terminal oxidase elicits reductive stress in mitochondria by which an electron would easily be released from the saturated electron transport chains. The released electron easily reduces molecular oxygen and generates the superoxide radical. The resulting superoxide might instantaneously react with NO, thereby inhibiting biological activity of this gaseous radical. Thus, cross-talk between superoxide and NO radicals might also play a critical role in the regulation of mitochondrial energy transduction (Fig. 4). M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95 93 Among various tissues, the rate of oxygen consumption is highest within the brain and, hence, this organ often becomes hypoxic. Thus, biological activity of NO in the brain would greatly be potentiated when cerebral concentrations of oxygen were decreased. This adaptive mechanism might appear to operate in order to compensate the decreased blood flow and energy production in ischemic brain at the levels of resistance arteries and neuronal mitochondria. Because aging of animals undergoes with the aging of blood vessels and cerebro-vascular injury is the major cause of death for aged people, oxidative stress evoked by the cross-talk between NO, superoxide and molecular oxygen might play a critical role in the maintenance of neurovascular function in the brain and other tissues. Such an oxidative stress might underlie the pathogenesis of age-related vascular injury and/or neuronal death during the long lifetime of aerobic organisms. Insights into the role of cross-talk between these oxyradicals should be further studied in order to understand the mechanism of human aging. Fig. 3. Oxygen dependent inhibition of mitochondrial respiration by NO. Nitric oxide inhibits state 3-respiration of mitochondria in a reversible manner. The inhibitory effect of NO increases with the decrease in oxygen tension. Kinetic analysis using specific inhibitors of electron transport system revealed that binding of NO to cytochrome c oxidase is responsible for the reversible inhibition. When electron transport chains of the inhibited mitochondria are fully reduced, one electron reduction of molecular oxygen is enhanced thereby increasing the rate of superoxide generation. 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